The creation of the null mutation for the c-Src proto-oncogene established that the expression of c-Src in osteoclasts is essential for osteoclast action and generation of ruffled borders and resorption lacunae. This molecule, and its upstream regulators and downstream substrate(s), represent the critical and central common pathway in osteoclastic bone resorption independent of how osteoclasts are stimulated. The major regulator of c-Src has recently been identified as c-Csk, a cytoplasmic tyrosine kinase which phosphorylates c-Src at Tyr 527 and renders c-Src inactive. However, c-Src is still phosphorylated at Tyr 527, even in Csk-deficient cells, suggesting the presence of other Csk-related kinases. These Csk-related kinases are distributed in a tissue- or cell-specific manner, implying that the Csk-related kinases may play a role in tissue-specific functions mediated by c-Src. The goals of the present application are to understand how Src protein expression and tyrosine kinase activity is regulated during osteoclastic bone resorption, and to identify Src-specific substrate(s). The Specific Aims are: 1) to determine the precise molecular mechanisms by which c-Src is regulated in osteoclasts, by (a) cloning and expressing the Csk-related kinases, (b) examining the effects of Csk-related kinases on c-Src activity, (c) examining the expression of the Csk-related kinases in osteoclasts, in vitro and in vivo, and determining the relationship between expression and subcellular localization of c-Src with Csk, Csk-related kinases and c-Src substrates in cells resorbing bone, (d) determining the function of the Csk-related kinases in osteoclasts by examining the effects of antisense oligodeoxynucleotides, and over-expression of intact and mutated dominant-negative Csk-related kinases, and (e) determining the effects of osteotropic factors on subcellular localization and tyrosine phosphorylation of c-Src, Csk, Csk-related kinases and c-Src substrates, and correlating these changes with effects on bone resorption; and 2) to identify the target substrate(s) specific for c-Src in osteoclasts, and unravel the downstream signaling pathways utilized in osteoclasts.